Digging Deep for Ancient Relics: A Survey of Protein Motifs in the Intergenic Sequences of Four Eukaryotic Genomes Running title: Survey of Protein Pseudomotifs in Intergenic Region ZhaoLei Zhang, Paul Harrison and Mark Gerstein*, † Department of Molecular Biophysics and Biochemistry and †Department of Computer Science, Yale University, 266 Whitney Avenue, New Haven, CT 06520-8114 *Corresponding author Tel: (203) 432 6105 Fax: (360) 838 7861 Email: [email protected] 1 Summary We have examined conserved protein motifs in the non-coding, intergenic regions ("pseudomotif patterns") and surveyed their occurrence in the fly, worm, yeast and human genomes (chromosomes 21 and 22 only). To identify these patterns, we masked out annotated genes, pseudogenes and repeat regions from the raw genomic sequence and then compared the remaining sequence, in six-frame translation, against 1319 patterns from the PROSITE database. For each pseudomotif pattern, the absolute number of occurrences is not very informative unless compared against a statistical expectation; consequently, we calculated the expected occurrence of each pattern using a Poisson model and verified this with simulations. Using a p-value cut-off of 0.01, we found 67 pseudomotif patterns over-represented in fly intergenic regions, 34 in worm, 21 in human and 6 in yeast. These include the Zinc finger, leucine zipper, nucleotide-binding motif and EGF domain. Many of the over-represented patterns were common to two or more organisms, but there were a few that were unique to specific ones. Furthermore, we found more over-represented patterns in the fly than in the worm, although fly has fewer pseudogenes. This puzzling observation can be explained by a higher deletion rate in the fly genome. We also surveyed under-represented patterns, finding 23 in the fly, 12 in worm, 18 in human and 2 in yeast. If intergenic sequences were truly random, we would expect an equal number of over and under-represented patterns. The fact that for each organism the number of over-represented patterns is greater than the number of under-represented ones implies that a fraction of the intergenic regions consist of ancient protein fragments that, due to accumulated disablements, have become unrecognizable to conventional techniques for gene and pseudogene identification. Moreover, we find that in aggregate the over-represented pseudomotif patterns occupy a substantial fraction of the intergenic regions. Keywords: genome, intergenic, pseudogene, protein motif, DNA loss 2 Introduction In the genomes of higher organisms, only a small portion encodes for protein sequences, and of the non protein-coding sequence only a small fraction is known to have regulatory functions. In the human genome, it is estimated that 97 % of the sequence are non-protein coding 1, 2. It has been a great challenge for biologists to understand the origin and possible functions of these intergenic sequences in the genome. Comparative alignment of non-coding sequences between human and mouse 3, 4, C. elegans and C. Briggsae 5, D. melanogaster and D. virilis 6 found that around 20 ~ 30% of the non-coding sequences in eukaryotic genomes are functionally constrained among evolutionally-related species, suggesting that these regions have important regulatory roles. Despite these progress, understanding the origin and function of intergenic sequences remains a final frontier in the post-genomic era. Pseudogenes are non-functional copies of functional genes in the genome; they are often referred to as “dead genes” 7, 8 in the sense that they have become disabled in the course of evolution as results of nucleotide substitutions, insertions or deletions. However these pseudogenes have not deviated too far from their original sequences, so they can still be recovered by homology matching and the observation of simple disablements in the sequence, such as premature stop codons or frame shifts. Large populations of pseudogenes have been discovered in the genomes of the worm 9, yeast 10 and human 11. It is logical to argue that there could be more ancient genes or gene fragments in the genome that, after becoming disabled and freed from selection pressure, have accumulated so many substitutions, insertions and deletions that they can no longer be recognized by homology matching methods. As an extension to our on-going research on pseudogenes 9-12, we would like here to survey the pseudogenic protein fragments that can be recovered from intergenic regions. In particular, comparing the occurrence of “living” and “dead” protein parts will directly address a number of interesting evolutionary questions: is the occurrence of a protein family in dead 3 proteins related to its occurrence in the live proteome? Or are there certain protein families that were more common in the past and are dying out of modern eukaryotic organisms? Do these decaying families have any similarity to proteins in supposedly “more primitive” bacteria and archea? Since these ancient gene relics or “pseudomotifs” used to be part of protein coding sequences, we decided to search for protein fragments in the amino acid sequences predicted from raw intergenic sequences. It is important to choose the appropriate searching queries. PROSITE 13, 14 is a database of biologically significant protein patterns and profiles; it is maintained and frequently updated, and every pattern in it represents a conserved protein motif 15. For each protein pattern from PROSITE, we did a comprehensive search in the intergenic region of the yeast, fly and worm genomes and the human chromosome 21 and 22, counting the number of occurrences of "pseudomotifs". Because of the small and fragmentary nature of many of the patterns, it is quite conceivable that many of the matches we found could have arisen purely by chance. Consequently, for a specific individual match, it is difficult to determine whether it was a real protein motif. However, the focus of our study is to survey the pattern occurrences in a collective and statistical way rather than on an individual basis. We designed a probabilistic framework so that for each PROSITE pattern we could calculate the expected number of occurrences based on a Poisson model, and consequently determine the statistical significance of the observed number of occurrences. Using a p-value cut-off of 0.01, we were able to find those patterns that have more than the expected number of occurrences (i.e., those that are over- represented) in the intergenic regions. We want to emphasize that we deleted all the easy matches, i.e. the genes, pseudogenes and repeats from the genome, and we still found significantly over-represented protein patterns. This suggests that at least a fraction of the intergenic sequences in the genome could have arisen from ancient protein-coding genes. 4 We like to consider our study as “genomic paleontology” because, just like paleontologists digging for animal fossils, we are searching for molecular fossils, i.e. ancient protein fragments. When paleontologists excavate a fossil site, it is likely that more recent and more complete fossil samples, perhaps a whole skeleton, would be found in the upper layer of the depository; this is analogous to the discovery of pseudogenes, which are more recent in evolution and bear more homology to functional genes. When the paleontologists dig deeper under the ground, they tend to find more ancient and more fragmented fossils (e.g. only an individual limb or a tooth), like our findings of ancient protein fragments. Results And Discussion Overall Many PROSITE Patterns are Over-represented We used a p-value cut-off of 0.01 to select patterns that have an “unusual” number of occurrences relative to expectation. We divided the 1319 PROSITE patterns into three groups: over-represented, under-represented, and similar-to-expectation. This last category was for patterns that have statistically non-significant differences in occurrence relative to expectation -- i.e. those that have p-values ≥ 0.01, regardless of occurrence. While we believe a p-value cut-off of 0.01 is stringent enough, we also analyzed our data with a stricter p-value cut-off of 0.001; our conclusions were unaffected. As can be seen in Table 1, we picked up some very strongly over-represented pseudomotif patterns, which have p-values as small as 1.0e-300. As summarized in Table 2 (A), the fly has the most over-represented patterns in its intergenic regions, the worm and the human (chromosome 21 and 22 only) have fewer and yeast has the least. The same trend can be seen for under-represented patterns as well. It is not surprising that for the majority of the PROSITE patterns, their occurrences are similar to expectation. The pseudomotif patterns we are looking for are very faint signals of ancient proteins; thus, it is expected the background noise level would be 5 significantly high. Furthermore, we have carefully removed all sources of known "signals" -- i.e. genes, pseudogenes, and repeats. It is interesting to estimate the fraction of the present intergenic region that once coded for ancient protein. If we multiply the length of each over-represented pseudomotif pattern by the difference between the observed and expected number of occurrences, we find that significantly over-represented patterns occupy ~3% of the total intergenic region (Table 4). On average a PROSITE motif is ~14 amino acids in size. This is 12% of the size (120) of an average protein domain 16, implying that the total coverage of the domains associated with the motifs is ~26 % of the intergenic region in all four genomes. Note that this result does not include the amount of intergenic DNA in "obvious" pseudogenes. Furthermore, it could be affected by the fact that the PROSITE database is an incomplete set of features and is heavily biased by human input. Nevertheless, based on our results it is reasonable to assume that at a significant part of the intergenic region, otherwise known as “junk DNA”, once were protein-coding genes.